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| Index Figure 1 Figure 2 Figure 3 Figure 4 | ||
| click
image for larger view (view larger image
at 100% magnification for best clarity) |
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Figure
1.
Visualization of Single mRNA Molecules in Living Mammalian Cells
(A) A schematic of the constructs used in this study. The cassettes expressing the MS2-GFP fusion protein and the reporter mRNAs are shown. (B and C) Visualization of the reporter mRNA with the MS2-GFP fusion protein. Cos cells transiently cotransfected with the pMS2-GFP and pRSV-Z-6-SV plasmids and hybridized in situ with a probe against the MS2 binding sites are shown. GFP, green; in situ, red; nuclei (DAPI), blue. (B) A cell expressing both the MS2-GFP fusion and the reporter showing a GFP signal in the cytoplasm colocalizing with the probe (yellow). The scale bar represents 20 µm. (C) A cell expressing the MS2-GFP fusion alone, showing only a nuclear signal. The scale bar represents 20 µm. (D) Improvement of the sensitivity of RNA detection. Cos cells transiently cotransfected with pMS2-GFP and pRSV-Z-24-SV and treated as in (B), except that images were deconvolved. GFP (green) and the in situ hybridization signal using probes to MS2 (red) colocalize in particles (yellow). The scale bar represents 2 µm. (E–G) Quantification of the number of RNA molecules per particle. Cos cells transiently cotransfected with pMS2-GFP and pGRE-Z-24-hGH. (E) Automated selection and analysis of the cytoplasmic GFP particles. The scale bar represents 10 µm. Left: acquired image. Right: deconvolved image with automatically selected objects that correspond to the GFP particles. (F) The histogram depicts the number of GFP molecules per particle. The results are from 8 cells (>600 particles). (G) The histogram depicts the number of mRNA molecules per GFP particle detected by in situ hybridization, using a single probe to LacZ. The results are from 5 cells (125 particles). |
